Pyrophosphate is a common product of biosynthetic reactions. Inorganic pyrophosphatase (PPase), also known as pyrophosphate phosphohydrolase, catalyzes hydrolysis of inorganic pyrophosphate (PPi) to two molecules of orthophosphate. PPase plays an vital role in RNA and DNA synthesis in vivo. By cleaving PPi, the enzyme shifts the overall equilibrium in favor of synthesis.
DNA polymerases catalyze the template-dependent incorporation of a deoxynucleotide onto the 3' hydroxyl terminus of a primer, with the concomitant release of inorganic pyrophosphatase (PPi). This polymerization reaction is reversible. DNA polymerases also catalyze the reverse reaction, pyrophosphorolysis, which is the degradation of DNA in the presence of PPi. The reaction is summarized below: EQU DNA.sub.n +dNTP.revreaction.DNA.sub.n+1 +PPi
In vitro nucleic acid amplification methods, such as the polymerase chain reaction (PCR), require DNA polymerization. PCR is described in U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188, each incorporated herein by reference. In each cycle of a PCR amplification, a double-stranded target sequence is denatured, primers are annealed to each strand of the denatured target, and the primers are extended by the action of a DNA polymerase. The process is repeated typically between 25 and 40 times. Initial amplification conditions are chosen which favor the forward (polymerization) reaction (high dNTP concentrations, low pyrophosphate concentration). However, the amplification reaction results in an accumulation of pyrophosphate which increases the rate of the reverse reaction (pyrophosphorolysis), thereby decreasing the overall efficiency of the amplification reaction.
Similarly, pyrophosphorolysis can be detrimental to DNA sequencing reactions. Accuracy in DNA sequencing reactions depends on precise band position, a decrease in size of only one nucleotide can result in gel artifacts such as diffuse or missing bands. Pyrophosphorolysis results in the removal of bases from the 3' end of the primer extension product. Furthermore, removal of the terminal ddNMP from a ddNMP-terminated fragment allows subsequent extension.
Thus, in both amplification and sequencing reactions, it is desirable to minimize the pyrophosphorolysis reaction. The addition of PPase to the reaction shifts the overall equilibrium in favor of synthesis by cleaving PPi. The use of PPase to improve sequencing reactions is described in Tabor and Richardson, 1990, J. Biol. Chem. 265(14):8322-8328; and in PCT Patent Publication No. WO 902111; both incorporated herein by reference. The use of PPase in to improve DNA synthesis by a DNA polymerase is described in PCT Patent Publication No. WO 94/05797, incorporated herein by reference.
Native PPase protein has been isolated from Thermus thermophilus and Thermus ruber cells (WO 94/05797). Purification of native protein is time consuming and labor intensive.